Technology Note pubs.acs.org/acsmedchemlett
Trifluoromethyl Fluorocoxib A Detects Cyclooxygenase‑2 Expression in Inflammatory Tissues and Human Tumor Xenografts Md. Jashim Uddin,* Brenda C. Crews, Imran Huda, Kebreab Ghebreselasie, Cristina K. Daniel, and Lawrence J. Marnett A. B. Hancock, Jr., Memorial Laboratory for Cancer Research, Department of Biochemistry, Chemistry and Pharmacology, Vanderbilt Institute of Chemical Biology, Center for Molecular Toxicology and Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, United States S Supporting Information *
ABSTRACT: Fluorocoxib A is an effective COX-2-targeted optical imaging agent, used for in vivo detection of inflammatory tissues and premalignant and malignant tumors that express elevated levels of COX-2 (Uddin et al. Cancer Res. 2010, 70, 3618− 3627). In an effort to discover novel optical probes for COX-2, a trifluoromethyl analogue of fluorocoxib A (CF3-fluorocoxib A) was synthesized and evaluated for its ability to inhibit COX-2 in vitro purified enzyme and human cancer cell lines. Kinetic analysis revealed that CF3-fluorocoxib A is a slow, tight binding inhibitor of COX-2 that exhibits low nanomolar inhibitory potency. While CF3-fluorocoxib A and fluorocoxib A are similar in structure, CF3-fluorocoxib A shows improved potency in inhibition of wtCOX-2 and with a series of site-directed COX-2 mutants. After intraperitoneal injection, selective uptake of CF3fluorocoxib A is detected in inflamed mouse paws compared to noninflamed contralateral paws by optical imaging, and uptake is blocked by pretreatment with the COX-2 inhibitor, celecoxib. Selective uptake is also detected in the COX-2-positive human tumor xenografts (1483 HNSCC) as compared with the COX-2-negative tumor xenografts (HCT116) in an in vivo nude mouse tumor model. These in vitro and in vivo studies suggest that binding to COX-2 is the major determinant of uptake of CF3fluorocoxib A into the inflamed tissues and tumor xenografts. Thus, this new COX-2-targeted imaging probe should find utility in the detection and evaluation of COX-2 status in naturally occurring malignancies. KEYWORDS: Cyclooxygenase-2 (COX-2), CF3-indomethacin, CF3-fluorocoxib A, optical imaging, inflammation, cancer
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visualization of COX-2 in inflammatory and malignant lesions.7 Herein, we report the synthesis and evaluation of a trifluoromethyl analogue of fluorocoxib A as a selective COX2 inhibitor in purified protein and cells. We also describe the enzyme−inhibitor binding kinetics and in vivo delivery of CF3fluorocoxib A to inflammatory tissues and human tumor xenografts (Figure 1). CF3-indomethacin was synthesized using a Fisher indole cyclization of 5,5,5-trifluorolevulinic acid lactone with 1-(4methoxyphenyl)-1-(4-chlorobenzoyl)hydrazine hydrochloride under acidic conditions, as described in a previous report.8
yclooxygenases (COX) catalyze the biotransformation of arachidonic acid into a wide variety of prostaglandins, which are important biological mediators of inflammation.1 COX-1 is constitutively expressed in most normal tissues, where it performs housekeeping functions, such as maintenance of vascular tone and cytoprotection of the gastric mucosa.2 COX-2 is an inducible enzyme that is expressed in inflammation, where it modulates edema and pain, and in proliferative diseases, where it promotes growth and enhances metastasis.3 COX-2 overexpression is an early event in tumorigenesis, and it plays a role in tumor progression.4 Selective COX-2 inhibitors are useful in the treatment of various cancers.5,6 Therefore, COX-2 can be used as a target for imaging of inflammation and cancer with fluorescently conjugated COX-2 inhibitors. We recently reported the synthesis and evaluation of fluorocoxib A for the selective © 2014 American Chemical Society
Received: November 26, 2013 Accepted: January 23, 2014 Published: January 23, 2014 446
dx.doi.org/10.1021/ml400485g | ACS Med. Chem. Lett. 2014, 5, 446−450
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Technology Note
Figure 1. Chemical evolution of COX-2-selective inhibitors from nonsteroidal anti-inflammatory drug indomethacin.
The CF3-indomethacin was then coupled with mono N-tertbutoxycarbonyl-butylenediamine using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole hydrate, and N,N-diisopropylethylamine to afford tbutyl 4-[2-{1-(4-chlorobenzoyl)-5-methoxy-2′-trifluoromethyl1H-indol-3-yl}acetamido]butylcarbamate (Scheme 1). Treat-
benzoyl)-5-methoxy-2′-trifluoromethyl-1H-indol-3-yl}acetamide (CF3 -fluorocoxib A) (Scheme 1). Synthetic procedures and the characterization of all the new compounds are described in the Supporting Information. CF3-fluorocoxib A was assayed against purified COX-2 or COX-1 by a previously reported thin layer chromatography assay.10 We found that CF3-fluorocoxib A is a selective and potent inhibitor of COX-2. The calculated LogP values of CF3fluorocoxib A and fluorocoxib A are 6.96 and 6.34, respectively. The IC50 value for inhibition of COX-2 is 0.56 μM with no inhibition of COX-1 up to 25 μM. CF3-fluorocoxib A was assayed in RAW264.7 murine macrophage-like cells to check for membrane permeability and subsequent COX-2 inhibition.11 The IC50 value for inhibition of COX-2 by CF3fluorocoxib A was 0.08 μM. Further, the ability of CF3fluorocoxib A to inhibit COX-2 in 1483 head and neck squamous cell carcinoma (HNSCC) cells was assayed.7 CF3fluorocoxib A was incubated with 1483 HNSCC cells at several concentrations (0−5 μM) for 30 min followed by the addition of 10 μM [1-14C]-arachidonic acid (∼55 mCi/mmol). CF3Fluorocoxib A inhibited COX-2 with an IC50 value of 0.59 μM. Nearly all COX inhibitors, whether nonselective, like indomethacin, or COX-2-selective, like celecoxib, bind with the enzyme in a noncovalent manner. The one notable exception is aspirin, which irreversibly inactivates COX-1 and COX-2 through covalent modification of an active site serine residue. Indomethacin and celecoxib are examples of slow, tight-binding COX inhibitors. They rapidly establish an equilibrium with a loosely bound enzyme−inhibitor complex, which slowly converts to a much more tightly bound complex (eq 1). Figure 2a shows the time- and concentration-dependent inhibition of mCOX-2 by CF3-fluorocoxib A. The timedependency of COX-2 inhibition is clearly evident, as it requires approximately 10 min to achieve maximal inhibition. Note that the plateau of 15% activity remaining at high CF3fluorocoxib A concentrations suggests some reversibility of the tightly bound enzyme−inhibitor complex. Figure 2b displays a plot of the observed single exponential rate constants for inhibition (k obs ) as a function of CF 3 -fluorocoxib A
Scheme 1a
a Reagents and conditions (a) H2N-(CH2)4-NH-BOC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole hydrate, N,N-diisopropylethylamine, dimethyl formamide, 25 °C, 16 h; (b) HCl (gas), CH2Cl2, 025 °C 1 h; (c) 5-carboxy-Xrhodamine N-succinimidyl ester, triethylamine, dimethylsulfoxide, 25 °C, 16 h.
ment of 4-[2-{1-(4-chlorobenzoyl)-5-methoxy-2′-trifluoromethyl-1H-indol-3-yl}acetamido]butylcarbamate with HCl (gas) gave N-(4-aminobutyl)-2-{1-(4-chlorobenzoyl)-5-methoxy-2′-trifluoromethyl-1H-indol-3-yl}acetamide hydrochloride. 5-Carboxy-X-rhodamine N-succinnimidyl ester (5-ROX NSE)9 was reacted with the free amine of N-(4-aminobutyl)-2{1-(4-chlorobenzoyl)-5-methoxy-2′-trifluoromethyl-1H-indol3-yl}acetamide hydrochloride in the presence of triethylamine to give N-{(5-carboxy-X-rhodaminyl)but-4-yl}-2-{1-(4-chloro447
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Technology Note
in dimethyl sulfoxide. After 3 h, the animals were lightly anesthetized with 2% isoflurane and placed in a Xenogen IVIS200 optical imaging system. CF3-fluorocoxib A targeted the inflamed footpad selectively over the contralateral control footpad (Figure 3a). The dependence of compound uptake on COX-2 in the inflammatory tissues was confirmed by blocking the COX-2 active site with celecoxib. We administered celecoxib dissolved in dimethyl sulfoxide (50 mg/kg, i.p.) at 24 h postcarrageenan and waited 1 h for absorption and blockage of the COX-2 active site prior to dosing with CF3fluorocoxib A (1 mg/kg, i.p.). At 3 h postinjection of CF3fluorocoxib A, we lightly anesthetized the animals with 2% isoflurane and imaged them using the Xenogen IVIS200 camera. There was no enrichment of CF3-fluorocoxib A in the inflamed paw compared to the control paw (Figure 3b). Figure 3c displays the relative uptake of CF3-fluorocoxib A in the inflamed footpad versus the control footpad by image analysis of the data in Figure 3a (n = 3, p = 0.02). Also, imaging was performed at 5 and 30 min points, where significant probe distribution was observed in both paws. We next evaluated the ability of CF3-fluorocoxib A to target COX-2 in human tumor xenografts. Female nude mice were injected in the left hip with COX-2-expressing human 1483 HNSCC cells or in the right hip with COX-2-null human colorectal carcinoma (HCT116) cells. The tumor xenografts were allowed to grow to approximately 800−1000 mm3. Animals were injected (1 mg/kg, i.p.) with CF3-fluorocoxib A. At 4 h postinjection, the animals were lightly anesthetized with 2% isoflurane and placed in the Xenogen IVIS 200 optical imaging system. A significant uptake of CF3-fluorocoxib A was documented in the COX-2-expressing 1483 tumors (Figure 4a), where as only a minimal uptake was observed in the COX2-null HCT116 tumors (Figure 4b). Figure 4c displays quantification of the uptake of CF3-fluorocoxib A in the 1483 and HCT116 tumors obtained from image analysis (n = 4, p = 0.01). This suggests that the difference in uptake of CF3fluorocoxib A in 1483 and HCT116 xenografts is due to their differential in COX-2 expression. A significant peritoneal accumulation was detected at the earlier time points, which is due the distribution of the CF3-fluorocoxib A in liver, kidney with clearance in both urine and feces. CF3−Fluorocoxib A exhibits promise for in vivo detection of COX-2-expressing tumors that are deep-seated, such as tumors located in bladder, colon or intestine using noninvasive endoscopic techniques. Although, CF3-fluorocoxib A and fluorocoxib A are similar in structure, dissimilarities or improved properties were observed for CF3-fluorocoxib A in inhibition assays with a series of sitedirected COX-2 mutants (Table 1).13 The compounds described earlier by our laboratory were conjugates of the nonselective NSAID, indomethacin, with fluorophores.7 The present compound is a conjugate of a COX-2-selective inhibitor, CF3-indomethacin, with a fluorophore. It possesses superior selectivity and binding characteristics for COX-2. The inhibition of COX-2 by fluorocoxib A is due to the key interactions of the 2′-CH3 group with the residues Ala-527, Val349, Ser-530, and Leu-531 that form a small hydrophobic pocket. Mutation of Val-349 to Ala increases the potency of fluorocoxib A by ∼2-fold due to enlargement of the pocket, whereas mutation to Leu reduces the pocket size and decreases the potency of fluorocoxib A by ∼6-fold. A more intense trend was observed for CF3-fluorocoxib A, where it showed an improved potency against Val-349 to Ala mutant and Val-349 to Leu mutant, suggesting that the 2′-trifluoromethyl group of
Figure 2. Kinetics of the time-dependent inhibition of COX-2 by CF3fluorocoxib A. The purified COX-2 enzyme was reconstituted with heme and preincubated with the inhibitor at 37 °C for various times (0, 0.5, 1, 3, 5, 10, 15, and 30 min) prior to the addition of the substrate (50 μM). (a) Time-dependent inhibition of COX-2 by CF3fluorocoxib A at the indicated concentrations. (b) Secondary plot of kobs versus inhibitor concentration used to generate values for KI, k2, and k−2.
concentration, which allows the determination of the equilibrium constant for initial association (KI = k−1/k1) and the forward and reverse rate constants for the conversion to the tightly bound enzyme−inhibitor complex (k2 and k−2, respectively, eq 2). CF3-fluorocoxib A and fluorocoxib A demonstrate similar affinities for initial complex formation, as indicated by their KI values (1.5 ± 0.35 and 1.7 ± 2.3 μM, respectively). The forward rate constants (k2) are also similar for these compounds (CF3-fluorocoxib A, k2 = 0.004 s−1; fluorocoxib A, k2 = 0.005 s−1), and the reverse rate constants (k−2) are essentially the same (CF3-fluorocoxib A, k−2 = 0.001 s−1; fluorocoxib A, k−2 = 0.001 s−1). The low rate constant for dissociation of the tightly bound complex is a strong contributing factor toward the potency of the compounds. k1
k2
k −1
k −2
E + I HooI [EI] HooI EI*
(1)
where KI = k−1/k1 and kobs =
k 2*[I] + k −2 KI + [I]
(2)
The C57BL/6 mouse footpad model is a well-established system for the study of COX-2-dependent inflammation. COX2-derived prostaglandins have a significant role in the acute edema induced by carrageenan injection into the paw.12 A major advantage of this inflammation model is the ability to image the inflamed mouse footpad in comparison to the noninflamed contralateral footpad without COX-2 expression. We injected 50 μL of 1% carrageenan in the rear right footpad of each mouse (body weight 20−25 g) and waited 24 h for inflammation to develop. Then, we injected the fluorescent CF3-fluorocoxib A (1 mg/kg) intraperitoneally (i.p.) dissolved 448
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Technology Note
Figure 3. continued lightly anesthetized with 2% isoflurane and imaged in the Xenogen IVIS 200 optical imaging system. There was minimal uptake of CF3fluorocoxib A in the inflamed footpad that was comparable to the noninflamed foot. (c) Quantitation of CF3-fluorocoxib A uptake in inflamed vs control footpad at 3 h postinjection of the agent (from data in panel a).
Figure 4. In vivo labeling of COX-2-expressing cancer by CF3fluorocoxib A. Female nude mice bearing COX-2-expressing 1483 HNSCC and COX-2-null HCT116 tumors were dosed with CF3fluorocoxib A (1 mg/kg, i.p.). The animals were imaged at 4 h postinjection of CF3-fluorocoxib A in a Xenogen IVIS200 optical imaging instrument. (a) A significant uptake of CF3-fluorocoxib A was documented in the COX-2-expressing 1483 tumors (arrow). (b) Minimal uptake of CF3-fluorocoxib A was documented in the COX-2negative HCT116 tumors. (c) Quantitation of CF3-fluorocoxib A uptake in COX-2-expressing 1483 tumors and COX-2-negative HCT116 tumors at 4 h postinjection of the agent.
Figure 3. In vivo labeling of COX-2 expression in C57BL/6 mouse footpad inflammation by CF3-fluorocoxib A. The inflammation was developed by injecting 50 μL 1% carrageenan into the right hind footpad. (a) At 24 h postcarrageenan, the mouse was injected with CF3-fluorocoxib A (1 mg/kg, i.p.). At 3 h postinjection of CF3fluorocoxib A, a mouse was lightly anesthetized with 2% isoflurane and imaged in the Xenogen IVIS 200 optical imaging system. A significant uptake of CF3-fluorocoxib A was documented in the inflamed footpad. (b) At 24 h postcarrageenan, the mouse was predosed with celecoxib (50 mg/kg, i.p.) 1 h before the administration of CF3-fluorocoxib A (1 mg/kg, i.p.). At 3 h postinjection of CF3-fluorocoxib A, the mouse was
CF3-fluorocoxib A binds with these residues more tightly then that of the 2′-methyl group of fluorocoxib A. Improved properties of CF3-fluorocoxib A were also observed with Ser530 to Ala mutant, Arg-120 to Ala mutant, or Val-89 to Trp/ Ser-119 to Trp double mutant, suggesting that the 2′449
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ACKNOWLEDGMENTS We are grateful to Dr. Carol Rouzer for critical reading and editing of this manuscript.
Table 1. Inhibition of Wild-Type Ovine COX-1, Wild-Type Mouse COX-2, and Mouse COX-2 Mutants by Fluorocoxib A and CF3-Fluorocoxib A wild-type or mutant enzymes wt hCOX-2 wt oCOX-1 V349A V349L S530A R120A V89W/S119W
fluorocoxib A (IC50 μM)a 0.70 >25 0.38 >4 0.32 2.00 1.30
CF3-fluorocoxib A (IC50 μM)a
REFERENCES
(1) Vane, J. R. Inhibition of Prostaglandin Synthesis as a Mechanism of Action for Aspirin-Like Drugs. Nat. New Biol. 1971, 231, 232−235. (2) Smith, W. L.; Garavito, R. M.; DeWitt, D. L. Prostaglandin Endoperoxide H Synthases (Cyclooxygenases)-1 and -2. J. Biol. Chem. 1996, 271, 33157−33160. (3) Li, G.; Yang, T.; Yan, J. Cyclooxygenase-2 Increased the Angiogenic and Metastatic Potential of Tumor Cells. Biochem. Biophys. Res. Commun. 2002, 299, 886−890. (4) Taketo, M. M. Cyclooxygenase-2 Inhibitors in Tumorigenesis (Part II). J. Natl. Cancer Inst. 1998, 90, 1609−1620. (5) Gupta, R. A.; DuBois, N. Cyclooxygenase-2 Inhibitor Therapy for the Prevention of Esophageal Adenocarcinoma in Barrett’s Esophagus. J. Natl. Cancer Inst. 2002, 94, 406−407. (6) Maier, T. J.; Schilling, K.; Schmidt, R.; Geisslinger, G.; Grosch, S. Cyclooxygenase-2 (COX-2)-Dependent and -Independent Anticarcinogenic Effects of Celecoxib in Human Colon Carcinoma Cells. Biochem. Pharmacol. 2004, 67, 1469−1474. (7) Uddin, M. J.; Crews, B. C.; Blobaum, A. L.; Kingsley, P. J.; Gorden, D. L.; McIntyre, J. O.; Matrisian, L. M.; Subbaramaiah, K.; Dannenberg, A. J.; Piston, D. W.; Marnett, L. J. Selective Visualization of Cyclooxygenase-2 in Inflammation and Cancer by Targeted Fluorescent Imaging Agents. Cancer Res. 2010, 70, 3618−3627. (8) Blobaum, A. L.; Uddin, M. J.; Felts, A. S.; Crews, B. C.; Rouzer, C. A.; Marnett, L. J. The 2′-Trifluoromethyl Analog of Indomethacin is a Potent and Selective COX-2 Inhibitor. ACS Med. Chem. Lett 2013, 4 (5), 486−490. (9) Uddin, M. J.; Marnett, L. J. Synthesis of 5- and 6-Carboxy-Xrhodamines. Org. Lett. 2008, 10, 4799−4801. (10) Kalgutkar, A. S.; Crews, B. C.; Rowlinson, S. W.; Marnett, A. B.; Kozak, K. R.; Remmel, R. P.; Marnett, L. J. Biochemically Based Design of Cyclooxygenase-2 (COX-2) Inhibitors: Facile Conversion of Nonsteroidal Antiinflammatory Drugs to Potent and Highly Selective COX-2 Inhibitors. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 925−930. (11) Kalgutkar, A. S.; Kozak, K. R.; Crews, B. C.; Hochgesang, G. P., Jr.; Marnett, L. J. Covalent Modification of Cyclooxygenase-2 (COX2) by 2-Acetoxyphenyl Alkyl Sulfides, a New Class of Selective COX-2 Inactivators. J. Med. Chem. 1998, 41, 4800−4818. (12) Meglio, P. D.; Ianaro, A.; Ghosh, S. Amelioration of Acute Inflammation by Systemic Administration of a Cell-Permeable Peptide Inhibitor of NF-κB Activation. Arthrit. Rheum. 2005, 52, 951−958. (13) Rowlinson, S. W.; Crews, B. C.; Lanzo, C. A.; Marnett, L. J. The Binding of Arachidonic Acid in the Cyclooxygenase Active Site of Mouse Prostaglandin Endoperoxide Synthase-2 (COX-2). A Putative L-Shaped Binding Conformation Utilizing the Top Channel Region. J. Biol. Chem. 1999, 274, 23305−23310.
0.56 >25 0.18 0.72 0.16 0.36 0.51
IC50 values are μM and represent time-dependent inhibition and average determinations from three experiments. a
trifluoromethyl group, the n-butyl diamide tether, or the 5-ROX fluorophore of CF3-fluorocoxib A interacts with the respective residues more tightly than the 2′-methyl group, the n-butyl diamide linker group, or the 5-ROX fluorophore group of fluorocoxib A. In summary, CF3-fluorocoxib A has been synthesized and evaluated as a potent fluorescent COX-2-specific inhibitor for optical imaging. The fluorescent CF3-fluorocoxib A inhibited COX-2 selectively in purified protein as well as in intact inflammatory and cancer cells. CF3-fluorocoxib A is a slow and tight binding inhibitor of COX-2 with similar binding kinetics for COX-2 as the parent fluorocoxib A. Although CF3fluorocoxib A and fluorocoxib A share similar structural features, CF3-fluorocoxib A is a more potent inhibitor of wtCOX-2 and of a series of COX-2 mutants. CF3-fluorocoxib A displays good selectivity of uptake in inflammatory tissues and COX-2-expressing tumors compared to control tissues or COX-2-negative tumors. Uptake of CF3-fluorocoxib A requires the expression of COX-2 at the target site. Uptake is reduced when the COX-2 active site is preblocked or in the absence of COX-2 expression in the target site. These in vitro and in vivo studies provide support for the conclusion that high specificity and tight binding to the COX-2 enzyme is the major determinant of uptake and retention of CF3-fluorocoxib A in inflamed tissues and tumors. Thus, CF3-fluorocoxib A represents a new optical imaging reagent for the detection and evaluation of COX-2 status in naturally occurring malignancies.
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Technology Note
ASSOCIATED CONTENT
S Supporting Information *
Full synthetic procedures and analytical and spectral characterization data of the synthesized compounds. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*(M.J.U.) Tel: 615-343-7326. Fax: 615-343-7534. E-mail: [email protected]. Funding
This work was supported by a grant from the National Institutes of Health and National Cancer Institute (CA136465, CA128323, CA128323-4, and CA128323-5). Notes
The authors declare no competing financial interest. 450
dx.doi.org/10.1021/ml400485g | ACS Med. Chem. Lett. 2014, 5, 446−450
Trifluoromethyl Fluorocoxib A Detects Cyclooxygenase‑2 Expression in Inflammatory Tissues and Human Tumor Xenografts Md. Jashim Uddin,* Brenda C. Crews, Imran Huda, Kebreab Ghebreselasie, Cristina K. Daniel, and Lawrence J. Marnett A. B. Hancock, Jr., Memorial Laboratory for Cancer Research, Department of Biochemistry, Chemistry and Pharmacology, Vanderbilt Institute of Chemical Biology, Center for Molecular Toxicology and Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, United States S Supporting Information *
ABSTRACT: Fluorocoxib A is an effective COX-2-targeted optical imaging agent, used for in vivo detection of inflammatory tissues and premalignant and malignant tumors that express elevated levels of COX-2 (Uddin et al. Cancer Res. 2010, 70, 3618− 3627). In an effort to discover novel optical probes for COX-2, a trifluoromethyl analogue of fluorocoxib A (CF3-fluorocoxib A) was synthesized and evaluated for its ability to inhibit COX-2 in vitro purified enzyme and human cancer cell lines. Kinetic analysis revealed that CF3-fluorocoxib A is a slow, tight binding inhibitor of COX-2 that exhibits low nanomolar inhibitory potency. While CF3-fluorocoxib A and fluorocoxib A are similar in structure, CF3-fluorocoxib A shows improved potency in inhibition of wtCOX-2 and with a series of site-directed COX-2 mutants. After intraperitoneal injection, selective uptake of CF3fluorocoxib A is detected in inflamed mouse paws compared to noninflamed contralateral paws by optical imaging, and uptake is blocked by pretreatment with the COX-2 inhibitor, celecoxib. Selective uptake is also detected in the COX-2-positive human tumor xenografts (1483 HNSCC) as compared with the COX-2-negative tumor xenografts (HCT116) in an in vivo nude mouse tumor model. These in vitro and in vivo studies suggest that binding to COX-2 is the major determinant of uptake of CF3fluorocoxib A into the inflamed tissues and tumor xenografts. Thus, this new COX-2-targeted imaging probe should find utility in the detection and evaluation of COX-2 status in naturally occurring malignancies. KEYWORDS: Cyclooxygenase-2 (COX-2), CF3-indomethacin, CF3-fluorocoxib A, optical imaging, inflammation, cancer
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visualization of COX-2 in inflammatory and malignant lesions.7 Herein, we report the synthesis and evaluation of a trifluoromethyl analogue of fluorocoxib A as a selective COX2 inhibitor in purified protein and cells. We also describe the enzyme−inhibitor binding kinetics and in vivo delivery of CF3fluorocoxib A to inflammatory tissues and human tumor xenografts (Figure 1). CF3-indomethacin was synthesized using a Fisher indole cyclization of 5,5,5-trifluorolevulinic acid lactone with 1-(4methoxyphenyl)-1-(4-chlorobenzoyl)hydrazine hydrochloride under acidic conditions, as described in a previous report.8
yclooxygenases (COX) catalyze the biotransformation of arachidonic acid into a wide variety of prostaglandins, which are important biological mediators of inflammation.1 COX-1 is constitutively expressed in most normal tissues, where it performs housekeeping functions, such as maintenance of vascular tone and cytoprotection of the gastric mucosa.2 COX-2 is an inducible enzyme that is expressed in inflammation, where it modulates edema and pain, and in proliferative diseases, where it promotes growth and enhances metastasis.3 COX-2 overexpression is an early event in tumorigenesis, and it plays a role in tumor progression.4 Selective COX-2 inhibitors are useful in the treatment of various cancers.5,6 Therefore, COX-2 can be used as a target for imaging of inflammation and cancer with fluorescently conjugated COX-2 inhibitors. We recently reported the synthesis and evaluation of fluorocoxib A for the selective © 2014 American Chemical Society
Received: November 26, 2013 Accepted: January 23, 2014 Published: January 23, 2014 446
dx.doi.org/10.1021/ml400485g | ACS Med. Chem. Lett. 2014, 5, 446−450
ACS Medicinal Chemistry Letters
Technology Note
Figure 1. Chemical evolution of COX-2-selective inhibitors from nonsteroidal anti-inflammatory drug indomethacin.
The CF3-indomethacin was then coupled with mono N-tertbutoxycarbonyl-butylenediamine using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole hydrate, and N,N-diisopropylethylamine to afford tbutyl 4-[2-{1-(4-chlorobenzoyl)-5-methoxy-2′-trifluoromethyl1H-indol-3-yl}acetamido]butylcarbamate (Scheme 1). Treat-
benzoyl)-5-methoxy-2′-trifluoromethyl-1H-indol-3-yl}acetamide (CF3 -fluorocoxib A) (Scheme 1). Synthetic procedures and the characterization of all the new compounds are described in the Supporting Information. CF3-fluorocoxib A was assayed against purified COX-2 or COX-1 by a previously reported thin layer chromatography assay.10 We found that CF3-fluorocoxib A is a selective and potent inhibitor of COX-2. The calculated LogP values of CF3fluorocoxib A and fluorocoxib A are 6.96 and 6.34, respectively. The IC50 value for inhibition of COX-2 is 0.56 μM with no inhibition of COX-1 up to 25 μM. CF3-fluorocoxib A was assayed in RAW264.7 murine macrophage-like cells to check for membrane permeability and subsequent COX-2 inhibition.11 The IC50 value for inhibition of COX-2 by CF3fluorocoxib A was 0.08 μM. Further, the ability of CF3fluorocoxib A to inhibit COX-2 in 1483 head and neck squamous cell carcinoma (HNSCC) cells was assayed.7 CF3fluorocoxib A was incubated with 1483 HNSCC cells at several concentrations (0−5 μM) for 30 min followed by the addition of 10 μM [1-14C]-arachidonic acid (∼55 mCi/mmol). CF3Fluorocoxib A inhibited COX-2 with an IC50 value of 0.59 μM. Nearly all COX inhibitors, whether nonselective, like indomethacin, or COX-2-selective, like celecoxib, bind with the enzyme in a noncovalent manner. The one notable exception is aspirin, which irreversibly inactivates COX-1 and COX-2 through covalent modification of an active site serine residue. Indomethacin and celecoxib are examples of slow, tight-binding COX inhibitors. They rapidly establish an equilibrium with a loosely bound enzyme−inhibitor complex, which slowly converts to a much more tightly bound complex (eq 1). Figure 2a shows the time- and concentration-dependent inhibition of mCOX-2 by CF3-fluorocoxib A. The timedependency of COX-2 inhibition is clearly evident, as it requires approximately 10 min to achieve maximal inhibition. Note that the plateau of 15% activity remaining at high CF3fluorocoxib A concentrations suggests some reversibility of the tightly bound enzyme−inhibitor complex. Figure 2b displays a plot of the observed single exponential rate constants for inhibition (k obs ) as a function of CF 3 -fluorocoxib A
Scheme 1a
a Reagents and conditions (a) H2N-(CH2)4-NH-BOC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole hydrate, N,N-diisopropylethylamine, dimethyl formamide, 25 °C, 16 h; (b) HCl (gas), CH2Cl2, 025 °C 1 h; (c) 5-carboxy-Xrhodamine N-succinimidyl ester, triethylamine, dimethylsulfoxide, 25 °C, 16 h.
ment of 4-[2-{1-(4-chlorobenzoyl)-5-methoxy-2′-trifluoromethyl-1H-indol-3-yl}acetamido]butylcarbamate with HCl (gas) gave N-(4-aminobutyl)-2-{1-(4-chlorobenzoyl)-5-methoxy-2′-trifluoromethyl-1H-indol-3-yl}acetamide hydrochloride. 5-Carboxy-X-rhodamine N-succinnimidyl ester (5-ROX NSE)9 was reacted with the free amine of N-(4-aminobutyl)-2{1-(4-chlorobenzoyl)-5-methoxy-2′-trifluoromethyl-1H-indol3-yl}acetamide hydrochloride in the presence of triethylamine to give N-{(5-carboxy-X-rhodaminyl)but-4-yl}-2-{1-(4-chloro447
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ACS Medicinal Chemistry Letters
Technology Note
in dimethyl sulfoxide. After 3 h, the animals were lightly anesthetized with 2% isoflurane and placed in a Xenogen IVIS200 optical imaging system. CF3-fluorocoxib A targeted the inflamed footpad selectively over the contralateral control footpad (Figure 3a). The dependence of compound uptake on COX-2 in the inflammatory tissues was confirmed by blocking the COX-2 active site with celecoxib. We administered celecoxib dissolved in dimethyl sulfoxide (50 mg/kg, i.p.) at 24 h postcarrageenan and waited 1 h for absorption and blockage of the COX-2 active site prior to dosing with CF3fluorocoxib A (1 mg/kg, i.p.). At 3 h postinjection of CF3fluorocoxib A, we lightly anesthetized the animals with 2% isoflurane and imaged them using the Xenogen IVIS200 camera. There was no enrichment of CF3-fluorocoxib A in the inflamed paw compared to the control paw (Figure 3b). Figure 3c displays the relative uptake of CF3-fluorocoxib A in the inflamed footpad versus the control footpad by image analysis of the data in Figure 3a (n = 3, p = 0.02). Also, imaging was performed at 5 and 30 min points, where significant probe distribution was observed in both paws. We next evaluated the ability of CF3-fluorocoxib A to target COX-2 in human tumor xenografts. Female nude mice were injected in the left hip with COX-2-expressing human 1483 HNSCC cells or in the right hip with COX-2-null human colorectal carcinoma (HCT116) cells. The tumor xenografts were allowed to grow to approximately 800−1000 mm3. Animals were injected (1 mg/kg, i.p.) with CF3-fluorocoxib A. At 4 h postinjection, the animals were lightly anesthetized with 2% isoflurane and placed in the Xenogen IVIS 200 optical imaging system. A significant uptake of CF3-fluorocoxib A was documented in the COX-2-expressing 1483 tumors (Figure 4a), where as only a minimal uptake was observed in the COX2-null HCT116 tumors (Figure 4b). Figure 4c displays quantification of the uptake of CF3-fluorocoxib A in the 1483 and HCT116 tumors obtained from image analysis (n = 4, p = 0.01). This suggests that the difference in uptake of CF3fluorocoxib A in 1483 and HCT116 xenografts is due to their differential in COX-2 expression. A significant peritoneal accumulation was detected at the earlier time points, which is due the distribution of the CF3-fluorocoxib A in liver, kidney with clearance in both urine and feces. CF3−Fluorocoxib A exhibits promise for in vivo detection of COX-2-expressing tumors that are deep-seated, such as tumors located in bladder, colon or intestine using noninvasive endoscopic techniques. Although, CF3-fluorocoxib A and fluorocoxib A are similar in structure, dissimilarities or improved properties were observed for CF3-fluorocoxib A in inhibition assays with a series of sitedirected COX-2 mutants (Table 1).13 The compounds described earlier by our laboratory were conjugates of the nonselective NSAID, indomethacin, with fluorophores.7 The present compound is a conjugate of a COX-2-selective inhibitor, CF3-indomethacin, with a fluorophore. It possesses superior selectivity and binding characteristics for COX-2. The inhibition of COX-2 by fluorocoxib A is due to the key interactions of the 2′-CH3 group with the residues Ala-527, Val349, Ser-530, and Leu-531 that form a small hydrophobic pocket. Mutation of Val-349 to Ala increases the potency of fluorocoxib A by ∼2-fold due to enlargement of the pocket, whereas mutation to Leu reduces the pocket size and decreases the potency of fluorocoxib A by ∼6-fold. A more intense trend was observed for CF3-fluorocoxib A, where it showed an improved potency against Val-349 to Ala mutant and Val-349 to Leu mutant, suggesting that the 2′-trifluoromethyl group of
Figure 2. Kinetics of the time-dependent inhibition of COX-2 by CF3fluorocoxib A. The purified COX-2 enzyme was reconstituted with heme and preincubated with the inhibitor at 37 °C for various times (0, 0.5, 1, 3, 5, 10, 15, and 30 min) prior to the addition of the substrate (50 μM). (a) Time-dependent inhibition of COX-2 by CF3fluorocoxib A at the indicated concentrations. (b) Secondary plot of kobs versus inhibitor concentration used to generate values for KI, k2, and k−2.
concentration, which allows the determination of the equilibrium constant for initial association (KI = k−1/k1) and the forward and reverse rate constants for the conversion to the tightly bound enzyme−inhibitor complex (k2 and k−2, respectively, eq 2). CF3-fluorocoxib A and fluorocoxib A demonstrate similar affinities for initial complex formation, as indicated by their KI values (1.5 ± 0.35 and 1.7 ± 2.3 μM, respectively). The forward rate constants (k2) are also similar for these compounds (CF3-fluorocoxib A, k2 = 0.004 s−1; fluorocoxib A, k2 = 0.005 s−1), and the reverse rate constants (k−2) are essentially the same (CF3-fluorocoxib A, k−2 = 0.001 s−1; fluorocoxib A, k−2 = 0.001 s−1). The low rate constant for dissociation of the tightly bound complex is a strong contributing factor toward the potency of the compounds. k1
k2
k −1
k −2
E + I HooI [EI] HooI EI*
(1)
where KI = k−1/k1 and kobs =
k 2*[I] + k −2 KI + [I]
(2)
The C57BL/6 mouse footpad model is a well-established system for the study of COX-2-dependent inflammation. COX2-derived prostaglandins have a significant role in the acute edema induced by carrageenan injection into the paw.12 A major advantage of this inflammation model is the ability to image the inflamed mouse footpad in comparison to the noninflamed contralateral footpad without COX-2 expression. We injected 50 μL of 1% carrageenan in the rear right footpad of each mouse (body weight 20−25 g) and waited 24 h for inflammation to develop. Then, we injected the fluorescent CF3-fluorocoxib A (1 mg/kg) intraperitoneally (i.p.) dissolved 448
dx.doi.org/10.1021/ml400485g | ACS Med. Chem. Lett. 2014, 5, 446−450
ACS Medicinal Chemistry Letters
Technology Note
Figure 3. continued lightly anesthetized with 2% isoflurane and imaged in the Xenogen IVIS 200 optical imaging system. There was minimal uptake of CF3fluorocoxib A in the inflamed footpad that was comparable to the noninflamed foot. (c) Quantitation of CF3-fluorocoxib A uptake in inflamed vs control footpad at 3 h postinjection of the agent (from data in panel a).
Figure 4. In vivo labeling of COX-2-expressing cancer by CF3fluorocoxib A. Female nude mice bearing COX-2-expressing 1483 HNSCC and COX-2-null HCT116 tumors were dosed with CF3fluorocoxib A (1 mg/kg, i.p.). The animals were imaged at 4 h postinjection of CF3-fluorocoxib A in a Xenogen IVIS200 optical imaging instrument. (a) A significant uptake of CF3-fluorocoxib A was documented in the COX-2-expressing 1483 tumors (arrow). (b) Minimal uptake of CF3-fluorocoxib A was documented in the COX-2negative HCT116 tumors. (c) Quantitation of CF3-fluorocoxib A uptake in COX-2-expressing 1483 tumors and COX-2-negative HCT116 tumors at 4 h postinjection of the agent.
Figure 3. In vivo labeling of COX-2 expression in C57BL/6 mouse footpad inflammation by CF3-fluorocoxib A. The inflammation was developed by injecting 50 μL 1% carrageenan into the right hind footpad. (a) At 24 h postcarrageenan, the mouse was injected with CF3-fluorocoxib A (1 mg/kg, i.p.). At 3 h postinjection of CF3fluorocoxib A, a mouse was lightly anesthetized with 2% isoflurane and imaged in the Xenogen IVIS 200 optical imaging system. A significant uptake of CF3-fluorocoxib A was documented in the inflamed footpad. (b) At 24 h postcarrageenan, the mouse was predosed with celecoxib (50 mg/kg, i.p.) 1 h before the administration of CF3-fluorocoxib A (1 mg/kg, i.p.). At 3 h postinjection of CF3-fluorocoxib A, the mouse was
CF3-fluorocoxib A binds with these residues more tightly then that of the 2′-methyl group of fluorocoxib A. Improved properties of CF3-fluorocoxib A were also observed with Ser530 to Ala mutant, Arg-120 to Ala mutant, or Val-89 to Trp/ Ser-119 to Trp double mutant, suggesting that the 2′449
dx.doi.org/10.1021/ml400485g | ACS Med. Chem. Lett. 2014, 5, 446−450
ACS Medicinal Chemistry Letters
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ACKNOWLEDGMENTS We are grateful to Dr. Carol Rouzer for critical reading and editing of this manuscript.
Table 1. Inhibition of Wild-Type Ovine COX-1, Wild-Type Mouse COX-2, and Mouse COX-2 Mutants by Fluorocoxib A and CF3-Fluorocoxib A wild-type or mutant enzymes wt hCOX-2 wt oCOX-1 V349A V349L S530A R120A V89W/S119W
fluorocoxib A (IC50 μM)a 0.70 >25 0.38 >4 0.32 2.00 1.30
CF3-fluorocoxib A (IC50 μM)a
REFERENCES
(1) Vane, J. R. Inhibition of Prostaglandin Synthesis as a Mechanism of Action for Aspirin-Like Drugs. Nat. New Biol. 1971, 231, 232−235. (2) Smith, W. L.; Garavito, R. M.; DeWitt, D. L. Prostaglandin Endoperoxide H Synthases (Cyclooxygenases)-1 and -2. J. Biol. Chem. 1996, 271, 33157−33160. (3) Li, G.; Yang, T.; Yan, J. Cyclooxygenase-2 Increased the Angiogenic and Metastatic Potential of Tumor Cells. Biochem. Biophys. Res. Commun. 2002, 299, 886−890. (4) Taketo, M. M. Cyclooxygenase-2 Inhibitors in Tumorigenesis (Part II). J. Natl. Cancer Inst. 1998, 90, 1609−1620. (5) Gupta, R. A.; DuBois, N. Cyclooxygenase-2 Inhibitor Therapy for the Prevention of Esophageal Adenocarcinoma in Barrett’s Esophagus. J. Natl. Cancer Inst. 2002, 94, 406−407. (6) Maier, T. J.; Schilling, K.; Schmidt, R.; Geisslinger, G.; Grosch, S. Cyclooxygenase-2 (COX-2)-Dependent and -Independent Anticarcinogenic Effects of Celecoxib in Human Colon Carcinoma Cells. Biochem. Pharmacol. 2004, 67, 1469−1474. (7) Uddin, M. J.; Crews, B. C.; Blobaum, A. L.; Kingsley, P. J.; Gorden, D. L.; McIntyre, J. O.; Matrisian, L. M.; Subbaramaiah, K.; Dannenberg, A. J.; Piston, D. W.; Marnett, L. J. Selective Visualization of Cyclooxygenase-2 in Inflammation and Cancer by Targeted Fluorescent Imaging Agents. Cancer Res. 2010, 70, 3618−3627. (8) Blobaum, A. L.; Uddin, M. J.; Felts, A. S.; Crews, B. C.; Rouzer, C. A.; Marnett, L. J. The 2′-Trifluoromethyl Analog of Indomethacin is a Potent and Selective COX-2 Inhibitor. ACS Med. Chem. Lett 2013, 4 (5), 486−490. (9) Uddin, M. J.; Marnett, L. J. Synthesis of 5- and 6-Carboxy-Xrhodamines. Org. Lett. 2008, 10, 4799−4801. (10) Kalgutkar, A. S.; Crews, B. C.; Rowlinson, S. W.; Marnett, A. B.; Kozak, K. R.; Remmel, R. P.; Marnett, L. J. Biochemically Based Design of Cyclooxygenase-2 (COX-2) Inhibitors: Facile Conversion of Nonsteroidal Antiinflammatory Drugs to Potent and Highly Selective COX-2 Inhibitors. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 925−930. (11) Kalgutkar, A. S.; Kozak, K. R.; Crews, B. C.; Hochgesang, G. P., Jr.; Marnett, L. J. Covalent Modification of Cyclooxygenase-2 (COX2) by 2-Acetoxyphenyl Alkyl Sulfides, a New Class of Selective COX-2 Inactivators. J. Med. Chem. 1998, 41, 4800−4818. (12) Meglio, P. D.; Ianaro, A.; Ghosh, S. Amelioration of Acute Inflammation by Systemic Administration of a Cell-Permeable Peptide Inhibitor of NF-κB Activation. Arthrit. Rheum. 2005, 52, 951−958. (13) Rowlinson, S. W.; Crews, B. C.; Lanzo, C. A.; Marnett, L. J. The Binding of Arachidonic Acid in the Cyclooxygenase Active Site of Mouse Prostaglandin Endoperoxide Synthase-2 (COX-2). A Putative L-Shaped Binding Conformation Utilizing the Top Channel Region. J. Biol. Chem. 1999, 274, 23305−23310.
0.56 >25 0.18 0.72 0.16 0.36 0.51
IC50 values are μM and represent time-dependent inhibition and average determinations from three experiments. a
trifluoromethyl group, the n-butyl diamide tether, or the 5-ROX fluorophore of CF3-fluorocoxib A interacts with the respective residues more tightly than the 2′-methyl group, the n-butyl diamide linker group, or the 5-ROX fluorophore group of fluorocoxib A. In summary, CF3-fluorocoxib A has been synthesized and evaluated as a potent fluorescent COX-2-specific inhibitor for optical imaging. The fluorescent CF3-fluorocoxib A inhibited COX-2 selectively in purified protein as well as in intact inflammatory and cancer cells. CF3-fluorocoxib A is a slow and tight binding inhibitor of COX-2 with similar binding kinetics for COX-2 as the parent fluorocoxib A. Although CF3fluorocoxib A and fluorocoxib A share similar structural features, CF3-fluorocoxib A is a more potent inhibitor of wtCOX-2 and of a series of COX-2 mutants. CF3-fluorocoxib A displays good selectivity of uptake in inflammatory tissues and COX-2-expressing tumors compared to control tissues or COX-2-negative tumors. Uptake of CF3-fluorocoxib A requires the expression of COX-2 at the target site. Uptake is reduced when the COX-2 active site is preblocked or in the absence of COX-2 expression in the target site. These in vitro and in vivo studies provide support for the conclusion that high specificity and tight binding to the COX-2 enzyme is the major determinant of uptake and retention of CF3-fluorocoxib A in inflamed tissues and tumors. Thus, CF3-fluorocoxib A represents a new optical imaging reagent for the detection and evaluation of COX-2 status in naturally occurring malignancies.
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Technology Note
ASSOCIATED CONTENT
S Supporting Information *
Full synthetic procedures and analytical and spectral characterization data of the synthesized compounds. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*(M.J.U.) Tel: 615-343-7326. Fax: 615-343-7534. E-mail: [email protected]. Funding
This work was supported by a grant from the National Institutes of Health and National Cancer Institute (CA136465, CA128323, CA128323-4, and CA128323-5). Notes
The authors declare no competing financial interest. 450
dx.doi.org/10.1021/ml400485g | ACS Med. Chem. Lett. 2014, 5, 446−450
